Streptavidin

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Streptavidin is a tetrameric protein purified from Streptomyces avidinii that binds very tightly to the vitamin biotin with a Kd of ~ 10-14 mol/L. This is one of the strongest biological and noncovalent interactions known, and is widely taken advantage of in scientific laboratories. One of the most common uses is for the purification of immunochemistries.

[edit] Uses in Biotechnology

The strong streptavidin-biotin bond can be used to "glue" various chemicals onto surfaces. One technique fixes DNA by first digesting DNA with a restriction exonuclease to produce either a blunt end, a 3' overhang or a 5' overhang. The DNA is then incubated with biotin-11-dUTP, a deoxyribonucleotide analog that is covalently attached to biotin, and the Klenow fragment of the holoenzyme DNA polymerase I of E. coli. The biotin-11-dUTP is incorporated into the 3' end of the strand complementary to the 5' ssDNA portion of the overhang. This is because DNA polymerases can only add nucleotides to the 3'-OH of DNA and not the 5'-phosphate.

  • 5'-ACTGGCTU-3'
  • 3'-TGACCGAACCGTT-5'

(where U is biotin-11-UTP incorporated into the DNA strand)

Assuming that care is taken to ensure that only one 5' overhang with only one possible site is available for dUTP incorporation, the result is a strand of DNA with a biotinylated end. One of the primary uses for biotinylated DNA is for binding (via non-covalent interactions) to streptavidin coated surfaces. With the DNA firmly attached to this substrate, various DNA hybridization and immunological assays can be performed. They may also be attached to streptavidin coated agarose microspheres, polystyrene or even paramagnetic beads. These complexes are most commonly used for the purification or isolation of DNA binding proteins. Other molecular biological techniques allow for exquisite control over DNA sequence, length, etc which make this a very powerful molecular tool.

[edit] References

  • Current Protocols in Protein Science (1998) 9.7-9.7.13
  • Zimmermann, R.M. et al, DNA stretching on functionalized gold surfaces. Nucleic Acids Research Vol. 22 No.3, Oxford University Press, (1994) pp. 492-497
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